The uptake of physiological nucleosides and their cytotoxic analogs in mammalian cells are mediated by nucleoside-specific transporters which accept a wide range of nucleosides as substrates. The transport mechanism in erythrocytes and many cultured cells is sensitive to nanomolar concentrations of the inhibitor p-nitro-benzylmercaptopurineriboside (NBMPR). These cells have high-affinity binding sites for NBMPR and occupancy of these sites by the inhibitor is correlated with loss of nucleoside transport activity. We have identified, in Walker 256 rat carcinoma cells, a second type of nucleoside transport activity which was relatively insensitive to this inhibitor. These cells had no high-affinity binding sites for NBMPR and transport was 1000-fold less sensitive to the inhibitor. A comparison of nucleoside transport in Walker 256 cells to that of NBMPR-sensitive S49 mouse lymphoma cells revealed little or no difference in substrate specificity or the kinetics of transport between the two mechanisms. Transport in the two cell lines did, however, differ in sensitivity to the sulfhydryl reagent p-mercuribenzenesulfonate, with Walker 256 cells 5-\to 10-fold more sensitive than S49 cells. Four other cell lines examined (L1210, P388, and L5178Y mouse leukemias and RPMI 6410 human lymphoblasts) all exhibited both NBMPR-sensitive and -insensitive nucleoside transport activity. In all aspects examined (kinetic constants for uridine transport, substrate specificity, and sensitivity to sulfhydryl reagents) the two transport mechanisms in L1210 cells were similar to the activity characterized in S49 and Walker 256 cells, respectively. Although the NBMPR-insensitive activity in these four cell lines was a small fraction (2 to 20%) of the total transport activity, it appeared to be an important determinant in the ability of NBMPR to block nucleoside uptake over prolonged periods (10 to 60 min). Studies in the coming year will focus on the identification of the plasma membrane proteins involved in the two transport processes in Walker 256, S49, and L1210 cells. (A)